Brillouin and Raman Micro-Spectroscopy: A Tool for Micro-Mechanical and Structural Characterization of Cortical and Trabecular Bone Tissues

Abstract

Human bone is a specialized tissue with unique material properties, providing mechanical support and resistance to the skeleton and simultaneously assuring capability of adaptation and remodelling. Knowing the properties of such a structure down to the micro-scale is of utmost importance, not only for the design of effective biomimetic materials but also to be able to detect pathological alterations in material properties, such as micro-fractures or abnormal tissue remodelling. The Brillouin and Raman micro-spectroscopic (BRmS) approach has the potential to become a first-choice technique, as it is capable of simultaneously investigating samples’ mechanical and structural properties in a non-destructive and label-free way. Here, we perform a mapping of cortical and trabecular bone sections of a femoral epiphysis, demonstrating the capability of the technique for discovering the morpho-mechanics of cells, the extracellular matrix, and marrow constituents. Moreover, the interpretation of Brillouin and Raman spectra merged with an approach of data mining is used to compare the mechanical alterations in specimens excised from distinct anatomical areas and subjected to different sample processing. The results disclose in both cases specific alterations in the morphology and/or in the tissue chemical make-up, which strongly affects bone mechanical properties, providing a method potentially extendable to other important biomedical issues.

Brillouin–Raman micro-spectroscopy and machine learning techniques to classify osteoarthritic lesions in the human articular cartilage

“…1 C evidences that the Brillouin spectra collected on both the cartilage and the bone tissues are characterized by at least two different peaks, namely P SOFT and P HARD , ranging between 4 and 13 GHz and 13 and 34 GHz respectively, with different relative ratios and average frequency shift going from one region to the other, confirming our previous findings in Refs. 26 – 28 . Their simultaneous presence in the same spectrum unveils the coexistence of micro-heterogeneities with different elastic moduli in the same scattering volume (for further details see “ Materials and methods ”) 27 , 29 , 32 .…”

“…In our previous works on the characterization of human healthy samples of bone and cartilage tissues, the detection of these two components was found to be dependent on the coexistence at the micrometric levels of an ordered phase of collagen bundles with different levels of mineralization and orientations (P HARD ), and a disordered phase of extracellular matrix, with poorly organized fibres, non-collagenous proteins and water, responsible for the cells survival and signalling inside the tissue (P SOFT ). Furthermore, the investigation of tissue organization in both the diaphysis and the epiphysis of femoral bones has revealed that also the bone marrow constituents—i.e., red cells and adipocytes—contribute significantly to both P SOFT intensity and frequency shift 27 , 28 .…”

“…However, the results obtained in this proof-of-principle will have to be further validated by a pre-clinical study considering (i) a more extended number of OA samples analyzed to develop an appropriate database of spectra, (ii) to extend the analyses on healthy samples collected from cadaver donors without signs of OA, increasing the inter-individual variability to have a comprehensive grading of the pathology, and (iii) to employ fresh/frozen sections to reproduce an environment more similar to the in-vivo condition. Concerning this, recent results obtained by our group 28 revealed that the paraformaldehyde (PFA) fixation, followed by the storage ethanol, can modify the local composition of the tissue due to the loss in its lipidic content so that the use of frozen samples likely will increase the efficacy of the algorithm, due to higher accessibility to the characterization cellular component. In the present study, it is worth noting that the same samples were first subjected to BRmS and then analysed by histological protocols.…”

“…In recent years, this technique has been successfully used to examine different biological entities—i.e., single-cells, tissue-phantom models based on biopolymeric hydrogels, microbial biofilms and tissues—and several pathological conditions, such as the corneal keratoconus, the amyloid plaques in Alzheimer's disease and pre-cancerous lesions in Barrett’s oesophagus 20 – 25 . Furthermore, in our previous works, we used BRamS for the characterization of musculoskeletal healthy tissue sections (i) belonging to different histological types and anatomical regions, such as the articular cartilage and both the cortical and trabecular bone tissues in the diaphyseal and the epiphyseal regions of the human femur in a healthy status 26 – 28 and (ii) treated with different sample processing, such as the freezing at − 80 °C and the paraformaldehyde fixation 29 .…”

Physicochemical Understanding of Biomineralization by Molecular Vibrational Spectroscopy: From Mechanism to Nature

“…For example, the structural information of the femoral shaft, the structural information at the joints, the spatial distribution of the skull, the trabecular structure could be readily obtained, etc. [52,165,216,217] The effects of different diseases on bone physicochemical properties were analyzed by Raman spectroscopy (Figure 6 ). It is observed that Raman spectroscopy can provide specific mechanical information about bones, predict the physiological age of organisms and the risk of fracture, etc., through the combination of logical operation and algorithm.…”